The genome sequence of the freshwater leech, Erpobdella octoculata (Linnaeus, 1758) (Hirudinida: Erpobdellidae)
Susan J. Skipp, Andrew Morrise, Jun Kim, Gonghua Lin, Thomas D. Lewin
TL;DR
This paper presents the genome sequence of the freshwater leech Erpobdella octoculata, providing a detailed genetic reference for this species.
Contribution
The study provides a high-quality, chromosome-level genome assembly for Erpobdella octoculata as part of a broader biodiversity initiative.
Findings
The genome assembly is 386.12 megabases long, with 98.81% scaffolded into 10 chromosomal pseudomolecules.
A mitochondrial genome of 19.12 kilobases was also assembled.
Gene annotation identified 19,465 protein-coding genes.
Abstract
We present a genome assembly from an individual Erpobdella octoculata (leeches; Annelida; Clitellata; Hirudinida; Erpobdellidae). The genome sequence has a total length of 386.12 megabases. Most of the assembly (98.81%) is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 19.12 kilobases. Gene annotation of this assembly on Ensembl identified 19 465 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
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Figure 5
Figure 6| Platform | PacBio HiFi | Hi-C |
|---|---|---|
|
| wkErpOcto2 | wkErpOcto1 |
|
| NHMUK014361447 | NHMUK014360727 |
|
| SAMEA7520983 | SAMEA7520811 |
|
| SAMEA7521083 | SAMEA7520867 |
|
| whole organism | head | terminal body |
|
| Sequel IIe | HiSeq X Ten |
|
| ERR12721044 | ERR12723455 |
|
| 1.86 million | 545.93 million |
|
| 22.86 Gb | 82.44 Gb |
|
| wkErpOcto2.1 |
|
| GCA_963978895.1 |
|
| GCA_963978845.1 |
|
| chromosome |
|
| 386.12 |
|
| 10 |
|
| 705 |
|
| 1.14 Mb |
|
| 101 |
|
| 39.24 Mb |
|
| Mitochondrion: 19.12 kb |
| INSDC
| Molecule | Length
| GC% |
|---|---|---|---|
| 1 | 52.90 | 34 | |
| 2 | 51.22 | 34.50 | |
| 3 | 43.52 | 34 | |
| 4 | 42.20 | 34.50 | |
| 5 | 39.24 | 34 | |
| 6 | 38.81 | 34 | |
| 7 | 32.92 | 34.50 | |
| 8 | 29.14 | 34.50 | |
| 9 | 26.46 | 34 | |
| 10 | 25.13 | 34.50 |
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (primary) | 6.C.Q51 | 6.C.Q40 |
| Contig N50 length | 1.14 Mb | ≥ 1 Mb |
| Scaffold N50 length | 39.24 Mb | = chromosome N50 |
| Consensus quality (QV) | Primary: 51.6; alternate: 53.0; combined: 51.6 | ≥ 40 |
|
| Primary: 79.25%; alternate: 78.38%; combined: 98.24% | ≥ 95% |
| BUSCO | C:86.7% [S:81.6%; D:5.1%]; F:6.5%; M:6.8%; n:954 | S > 90%; D < 5% |
| Percentage of assembly
| 98.81% | ≥ 90% |
- —Wellcome Trust
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Taxonomy
TopicsLeech Biology and Applications · Invertebrate Taxonomy and Ecology · Diatoms and Algae Research
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinea; Hirudinida; Erpobdelliformes; Erpobdellidae; Erpobdella; Erpobdella octoculata (Linnaeus, 1758) (NCBI:txid39305)
Background
Erpobdella octoculata (Linnaeus, 1758) ( Figure 1) is a common and widespread freshwater leech in Europe. It is found throughout Britain and Ireland, but is relatively uncommon in northern Scotland. It occurs in many freshwater habitats and can be abundant in organically enriched streams ( Murphy & Learner, 1982).
Photograph of Erpobdella octoculata by WWalas (not the specimen used for genome sequencing).
Field gut-content studies of E. octoculata show that its diet is dominated by chironomid larvae and oligochaetes ( Beracko & Rogánska, 2014). It is a macrophagous predator that often swallows invertebrate prey whole, especially chironomid larvae. It also sucks fluids from wounded or dead invertebrates and will scavenge vertebrate carrion. Newly hatched juveniles feed by sucking fluids from wounded prey ( Kutschera, 2003).
Life history of E. octoculata varies along stream gradients. Upstream populations are usually biennial and iteroparous; downstream they are annual and semelparous with faster growth and higher reproductive effort ( Beracko & Rogánska, 2014).
We report a chromosomally complete genome sequence for Erpobdella octoculata, produced as part of the Darwin Tree of Life project. This is the first whole genome sequence for the family Erpobdellidae as of October 2025 (data obtained via NCBI datasets, O’Leary et al., 2024).
Methods
Sample acquisition
The specimen used for genome sequencing was an adult Erpobdella octoculata (specimen ID NHMUK014361447, ToLID wkErpOcto2), collected from River Weaver Hunts Lock, UK (latitude 53.251, longitude –2.5189) on 2019-03-18. A second specimen was used for Hi-C sequencing (specimen ID NHMUK014360727, ToLID wkErpOcto1), collected from Shropshire Union Canal, Bagley Lane, UK (latitude 52.9754, longitude –2.5097) on 2019-03-18. Sample metadata were collected in line with the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
Nucleic acid extraction
Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). The wkErpOcto2 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the whole organism was homogenised by powermashing using a PowerMasher II tissue disruptor. HMW DNA was extracted using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 15.0 ng/μL and a yield of 6 000.00 ng.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), following the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using a Qubit Fluorometer v4.0 (ThermoFisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.
The sample was sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, and to perform primary and secondary analysis of the data upon completion.
Hi-C
** Sample preparation and crosslinking **
The Hi-C sample was prepared from 20–50 mg of frozen head and terminal body tissue of the wkErpOcto1 sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
** Hi-C library preparation and sequencing **
Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Library amplification was performed using KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).
Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again and equimolar and/or weighted 2.8 nM pools were created. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Sequencing was performed using paired-end 150 bp reads on the HiSeq X Ten.
Genome assembly
Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.
The HiFi reads were assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded in YaHS ( Zhou et al., 2023) with the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. TreeVal was used to generate the flat files and maps for use in curation. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Manual corrections included 57 breaks, 54 joins, and removal of 20 haplotypic duplications. This reduced the scaffold count by 13.6%, reduced the scaffold N50 by 1.6%, and reduced the total assembly length by 2.6%. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.
Assembly quality assessment
The Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The genome was analysed using the BlobToolKit pipeline, a Nextflow implementation of the earlier Snakemake version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
Genome sequence report
Sequence data
PacBio sequencing of the Erpobdella octoculata specimen generated 22.86 Gb (gigabases) from 1.86 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 370.71 Mb, with a heterozygosity of 1.10% and repeat content of 39.78% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 58× coverage. Hi-C sequencing produced 82.44 Gb from 545.93 million reads, which were used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Frequency distribution of k-mers generated using GenomeScope2.The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The final assembly has a total length of 386.12 Mb in 101 scaffolds, with 604 gaps, and a scaffold N50 of 39.24 Mb ( Table 2).
Most of the assembly sequence (98.81%) was assigned to 10 chromosomal-level scaffolds. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). There are a number of heterozygous inversions at the following locations: Chromosome 1 ~50.5–52 Mbp, Chromosome 2 ~23.13–24.33 Mbp, Chromosome 2 ~32.6–33.9 Mbp, Chromosome 3 ~17.5–18.8 Mbp, Chromosome 4 ~33.64–36.02 Mbp, Chromosome 10 ~11.11–12.07.
Hi-C contact map of the Erpobdella octoculata genome assembly. Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.
Table 3.: Chromosomal pseudomolecules in the primary genome assembly of Erpobdella octoculata wkErpOcto2.
The mitochondrial genome was also assembled (length 19.12 kb, OZ022248.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
The combined primary and alternate assemblies achieve an estimated QV of 51.6. The k-mer completeness is 79.25% for the primary assembly, 78.38% for the alternate haplotype, and 98.24% for the combined assemblies ( Figure 4).
Evaluation of k-mer completeness using MerquryFK.This plot illustrates the recovery of k‐mers from the original read data in the final assemblies. The horizontal axis represents k‐mer multiplicity, and the vertical axis shows the number of k‐mers. The black curve represents k‐mers that appear in the reads but are not assembled. The green curve corresponds to k‐mers shared by both haplotypes, and the red and blue curves show k‐mers found only in one of the haplotypes.
BUSCO v.5.5.0 analysis using the metazoa_odb10 reference set ( n = 954) identified 86.7% of the expected gene set (single = 81.6%, duplicated = 5.1%). The snail plot in Figure 5 summarises the scaffold length distribution and other assembly statistics for the primary assembly. The blob plot in Figure 6 shows the distribution of scaffolds by GC proportion and coverage.
Assembly metrics for wkErpOcto2.1.The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1 000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the metazoa_odb10 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
BlobToolKit GC-coverage plot for wkErpOcto2.1.Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.
Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the primary assembly, is 6.C.Q51, meeting the recommended reference standard.
Table 4.: Earth Biogenome Project summary metrics for the Erpobdella octoculata assembly.
Genome annotation report
The Erpobdella octoculata genome assembly (GCA_963978895.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 30 689 transcribed mRNAs from 19 465 protein-coding and 4 219 non-coding genes. The average transcript length is 5 597.31 bp, with an average of 1.30 coding transcripts per gene and 5.50 exons per transcript. For further information about the annotation, please refer to the Ensembl annotation page.
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission of materials by a Darwin Tree of Life Partner is subject to the ‘Darwin Tree of Life Project Sampling Code of Practice’, which can be found in full on the Darwin Tree of Life website. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project. Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. The overarching areas of consideration are:
Ethical review of provenance and sourcing of the materialLegality of collection, transfer and use (national and international)
Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances, other Darwin Tree of Life collaborators.
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